The beta-lactam antibiotics are the most heavily prescribed antibacterial chemotherapeutic agents in clinical use today. A key structural feature of all these antibiotics is the beta-lactam, a four-membered ring in which a carbonyl and nitrogen are joined in an amide linkage. Beta-Lactamases (beta- lactamhydrolases, EC 3. 5. 2. 6) are enzymes produced by pathogenic bacteria which allow them to become resistant to beta-lactam antibiotics such as penicillins and cephalosporins. These enzymes efficiently catalyze amide hydrolysis of the beta-lactam rings in these antibiotics thereby destroying their bactericidal activity. Mechanistic studies of beta- lactamases are important in that they can provide information useful in the development of beta-lactamase inhibitors or new, more effective antibiotics. One important class of beta-lactamases is metal-dependent. This proposal describes a study of the metallo-beta-lactamases produced by the bacterium Bacillus cereus. The experimental approach is multidisciplinary in nature in that it involves the use of a combination of recombinant DNA technology, site-directed mutagenesis, protein chemistry, enzymology, and electronic, circular dichroic (CD) and time-resolved electron paramagnetic resonance (EPR) spectroscopy to examine this important class of metalloenzymes.